Mammalian bone tissue comprises a number of proteins, including structural proteins such as collagen as well as osteogenic proteins that induce or promote bone growth. Recognition of the existence of osteogenic proteins in bone tissue has led to the discovery of a family of protein molecules known as the Bone Morphogenetic Proteins (BMPs). BMPs are members of the TGF-β superfamily of proteins, which includes additional proteins that provide tissue-inductive responses in vivo, including TGF-β1, TGF-β2, and TGF-β3. Structures for proteins designated BMP-1 through BMP-18 have been isolated and additional related proteins may be found. The unique inductive activity of the BMPs, along with their presence in bone tissue, suggests that they are involved in the regulation of bone repair processes and possibly in the normal maintenance of healthy bone tissue. There is a great need for such proteins for the induction and/or augmentation of bone growth following surgical bone repair or reconstruction procedures in human and animal patients.
Much research has been directed to producing, either by recombinant DNA techniques or by purification of naturally occurring proteins, specific osteoinductive proteins and protein mixtures. Protein mixtures having BMPs and other inductive proteins may be isolated from bone tissue according to known procedures. One of the earliest such procedures is disclosed in U.S. Pat. No. 4,294,753 to Urist, which provides a process for isolating bone proteins from bone tissue by demineralizing the bone tissue in acid. The demineralized collagen bone matrix is reduced to gelatin by adding a mineral salt. Osteoinductive BMPs are extracted from the gelatin using a solubilizing agent, such as guanidine hydrochloride and/or urea. The solubilized proteins are then purified by dialysis and several washing steps.
The processes disclosed in the '753 patent are also inherently wasteful. The demineralization step—the first step in the BMP isolation procedure—involves contacting the bone with hydrochloric acid to dissolve the mineral components of the bone and separate them from the protein components. The mineralized acid medium is discarded. Because BMPs are soluble in acids, a significant fraction of the BMPs are immediately lost at the beginning of processing.
The chemical reagents used to solubilize and extract the osteogenic proteins from the demineralized bone in the '753 procedures, i.e., guanidine hydrochloride (GuHCl) and urea, are cytotoxic. Consequently, the bone proteins must be subjected to extensive and time-consuming purification procedures to ensure that the BMPs obtained by the isolation procedures are free of cytotoxic agents and remain osteogenic when administered to the patient.
U.S. Pat. No. 4,619,989, also to Urist, discloses an improved process for isolating BMPs that involves additional dialysis purification steps beyond those disclosed in the '753 patent. Such steps increase still further the time required to isolate usable BMP mixtures. In addition, the additional purification steps further reduce protein yield and, worse still, may remove BMP fractions that are either osteogenic per se or have a synergistic effect with the remaining BMP proteins.
An improved method of isolating and purifying BMP-containing mixtures is described in U.S. Pat. Nos. 5,290,763 and 5,371,191. Both the '763 and '191 patents disclose a multistep process to provide highly purified BMP-containing mixtures. The process involves demineralization, protein extraction, high and low molecular weight ultrafiltration steps, an anion exchange process, a cation exchange process, and a reverse-phase HPLC process. Although the resulting BMP-containing mixture is highly osteogenic, the process is lengthy, requires expensive equipment, and has low yields.
To be effective as an osteogenic agent, BMPs must be delivered to a site within the body and retained in place for a period of weeks or months. The '753 patent discloses the co-precipitation of BMPs with a calcium salt, such as calcium carbonate, calcium silicate or calcium oxalate. An improved matrix material for release of BMPs is disclosed in U.S. Pat. No. 4,596,574, also to Urist. The disclosed matrix comprises biodegradable porous ceramic material, such as tricalcium phosphate. The BMPs may be deposited in the pores of the ceramic material by immersing the ceramic material in a solution containing the BMPs and lyophilizing the solution away. According to the '574 patent, the BMP-loaded ceramic resulted in substantial additional bone growth for a given dosage, and also lowered the required threshold dose for inducing bone growth.
Another BMP and carrier matrix product is provided in U.S. Pat. No. 5,563,124 to Damien et al. The '124 patent discloses a carrier comprising calcium carbonate, specifically aragonite, to which a BMP mixture, such as the mixtures disclosed in U.S. Pat. No. 5,290,763, is added. The BMPs may be added to the carrier by applying a solution of the BMPs to the aragonite carrier and then lyophilizing the solution. Alternatively, calcium carbonate particulates can be mixed with a matrix such as collagen, fibrin or alginate dispersion to form a composite, with the BMP solution added after drying the composite. In a still further embodiment, BMPs in solution may be added to a dispersion, which is then mixed with particulate calcium carbonate and dried.
There remains a need for BMP mixtures that may be easily, quickly and economically isolated from bone tissue in high yields, promote rapid osteoinduction when implanted in a human or animal patient, and that are amenable to combination with a wide variety of carriers.
It is an object of the present invention to provide a rapid, economical process for obtaining osteogenic BMP mixtures from mammalian bone tissue.
It is another object of the present invention to provide processes for recovering osteogenic BMPs from bone demineralization waste streams.
It is another object of the invention to provide a process for obtaining osteogenic BMP mixtures in high yields from mammalian bone tissue.
It is a still further object of the invention to provide a method of isolating osteogenic BMP mixtures from mammalian bone tissue that minimizes loss of BMPs from the bone tissue source.
It is a further object of the invention to provide a method of isolating osteogenic BMP mixtures from mammalian bone tissue that minimizes or avoids altogether the use of time-consuming dialysis procedures.
It is a further object of the invention to provide protein mixtures prepared by the foregoing processes.
It is a further object of the invention to provide implantable devices comprising a mixture of BMPs isolated from mammalian bone tissue.